Reversed-phase chromatography of proteins and peptides on compressed continuous beds

Abstract

The polymer beds described are synthesized in aqueous solution directly in the column or batchwise in the form of large clusters of small particles. The conventional, expensive step involving prepreparation of beads in an organic solvent is thus omitted. Beds were synthesized from piperazine diacrylamide, methacrylamide and allyl glycidyl ether. The epoxy-activated beds thus obtained were used for covalent attachment of either nonpolar ligands (e.g. octadecanol) or polar OH-rich substances (e.g. dextran). The non-polar beds were used for reversed-phase chromatography, as were polar ones following coupling with 1,2-epoxyoctadecane. Coating with OH-rich substances serves two purposes: (I) the matrix becomes hydrophilic, decreasing nonspecific interactions (modifiers can be excluded) and hence increases resolution and (II) many—OH groups are available (e.g. for coupling to epoxides), a prerequisite for high ligand density. Resolution of proteins was high even at high flow rates. Depending on the method used for the synthesis of the bed, resolution of proteins either increased with an increase in flow rate or decreased slinghtly. Choice of the correct temperature was very important for high resolution of CNRr-digested peptides.